By Daniel Herschlag
This MIE quantity offers laboratory suggestions that goal to foretell the constitution of a protein which could have super implications starting from drug layout, to mobile pathways and their dynamics, to viral access into cells. specialist researchers introduce the main complicated applied sciences and strategies in protein constitution and foldingIncludes strategies on tiling assays.
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Extra resources for Biophysical, Chemical and Functional Probes of RNA Structure, Interactions and Folding: Interaction and Folding
Pool the fractions corresponding to the clean RNA oligonucleotide and concentrate using 15 ml Centriprep centrifugal devices with 10 kDa MWCO-cutoff for RNAs larger than 30 nt or 3 kDa cutoff for smaller RNA oligonucleotides. Concentrate the RNA samples to 1 ml and then equilibrate them into the appropriate buffer using three to five consecutive 15 ml buffer exchanges. Concentrate the RNA to 1 ml at each step to maximize the efficiency of the buffer exchange (two spins) and then thoroughly mix with 14 ml fresh buffer for another round of centrifugation.
2007). In Section 6, we briefly present representative data from single molecule Fo¨rster resonance energy transfer (smFRET) experiments conducted on dye-labeled RNA molecules prepared by splinted RNA ligation. 2. General Considerations for Splinted RNA Ligation RNA ligation approaches typically employ one of several naturally occurring RNA ligase enzymes to catalyze the ATP-dependent joining of a ‘‘donor’’ fragment possessing a 50 -monophosphate and an ‘‘acceptor’’ fragment terminating in a 30 -hydroxyl group.
27 28 Benjamin M. Akiyama and Michael D. Stone limitation ($ 70 bases). Consequently, experimental approaches involving specific chemical modifications of larger RNAs require the use of RNA ligation methods. The aim of this chapter is to describe a general approach for covalently joining multiple site-specifically modified RNA fragments, drawing from our fluorescence-based structural studies of telomerase RNA as an example. 1. Introduction The essential role of RNA as a cellular messenger of genetic information has been well appreciated since the early 1960s.